Many different methods for the fractionation of subcellular organelles have been reported. However, no protocol for rapid separation of plasma membrane, Golgi/trans-Golgi network (TGN) and endosomes is available to date. Such a method is a prerequisite for a quantitative biochemical analysis of vesicular transport from the Golgi/TGN compartment to plasma membrane and endosomes. Here a density gradient electrophoresis protocol is described that allows the fractionation of these organelles in one step. This protocol requires only low-cost instrumentation available in most biochemical laboratories.